Abstract Pathogenic isolates were collected from different ricecropping regions in southern China to dissect the pathogenic disintegration and variation of Xanthomonas oryzae pv. oryzae （Xoo）. Two sets of riceXoo differential hosts， including Chinese system with such five cultivars as IR26， Java14， Nangeng15， Tetep and Jingang 30 and international system with a series of nearisogenic lines （NILs） including IRBB5， IRBB13， IRBB3， IRBB14， IRBB2 and IR24 carrying different known resistance genes，were used to detect pathogenic disintegration for the reaction between host and pathogen with leafclippingmethod at the rice booting stage. The results showed the type of the pathogen were divided intosix pathotypes， i.e.， I， II， III， IV， V and IX based on the Chinese differential system， and seven pathogenicraces including R1， R2， R3， R4， R5， R8 and R10 based on the international differential system. The pathogenicity frequency of Xoo pathotypes V and IV and pathogenic races R8 and R5 were 27.40%， 19.30% and 44.67%， 15.34%， which were considered to be the prevailing races in southern China.Pathogenic rates of pathotypes IV， V， IX and races R8， R5 against 500 varieties derived from southernChina were 96.40%， 95.00% ， 50.40%， 62.00%， and 42.60%， respectively. Among which pathotype IX was the most virulent pathotype. The pathotype V became preponderant pathotype and the new pathotype IX grew up quickly.
　　Key words Xanthomonas oryzae pv. oryzae； Differential variety； Pathotype； Race
　　Received： August 8， 2017 Accepted： November 12， 2017
　　Supported by Special Fund of Agroscientific Research in Public Interest （201303015）； Earmarked Fund for China Agriculture Research System （CARS0124）； Natural Science Foundation of China （31272010）.
　　Shen CHEN （1978-）， male， P. R. China， associate professor， devoted to research about disease resistanceand genetics.
　　*Corresponding author， Email： 2244642820@qq.com
　　Bacterial blight is an important disease in the southern part of China， which seriously threatens the safe production of rice. Because the pathogen of this disease harms rice plants within vascular bundles， there is still no safe efficient systemic bactericide， and chemical prevention is ineffective. Furthermore， the Xanthomonas oryzae pv. oryzae （Xoo） infects injured parts mainly through running water， and mechanical injury caused by typhoon rainstorm and flood create favorable conditions for its epidemic outbreak with the characteristics of acute occurrence， fast propagation and difficult control[1]. The control of this disease with resistant varieties is the most effective and harmless choice， while the selection of corresponding resistant gene directing at the pathogenicity of Xoo and its variability is a basis for the breeding for disease resistance， as well as a key link for the application of resistant varieties. Therefore， the accurate detection of the disintegration and variation of the pathogenicity of Xoo and the continuous monitoring of its development dynamics has very important scientific significance and application value to the analysis of variety resistance， the prediction of the occurrence and epidemic trend of the disease， the exploration and utilization of effective resistant gene and the formulation of the strategic decisions for the effective control of bacterial blight. 　　Certain progress was once made on the control of bacterial blight using the resistance in the host， but in recent years， due to the variation of the pathogenicity of Xoo， the variety resistance was lost， accompanied by the harm running wild[2]. There are many studies about the disintegration and variation of the pathogenicity of Xoo. Kuhara et al.[3] first found the difference in the pathogenicity of Xoo. Ezuka & Horiho[4] divided Xoo in Japan into five populations. Then， main riceproducing countries in Southeast Asia including the Philippines， Indonesia and Thailand conducted researches on the disintegration of the pathogenicity of Xoo， and also found different pathotypes and races[5]. Fang et al.[6] indentified the bacterial lines through the unified identification of the hosts in China （Jingang 30， Tetep， Nanjing 15， Java14 and IR26）， and seven pathotypes were divided， i.e.， pathotypes I， II， III， IV， V， VI and VII. Wang et al.[7] reported eight pathotypes， seven of which accords with the unified identification of bacterial lines in China， and the last one is a new pathotype with weaker pathogenicity found in Yunnan. Xu et al.[8] selected several nearisogenic lines bred by International Rice Research Institute， and combined with the differential hosts composed of partial varieties among Chinese differential hosts， the isolates of rice bacterial blight were divided into eight races. Zeng et al.[9] monitored the pathotypes of Xoo in Guangdong， and found that the tested isolates reacted in the new mode SSSSS on Chinese differential hosts. Yang et al.[10] selected nearisogenic differential hosts and divided all hosts in China into nine races. Que et al.[11] divided the isolates of Xoo in northern China into 16 races. Chen et al.[12] determined 29 isolates in Guangxi， which all exhibited the strongly virulent SSSSS mode on Chinese differential hosts.
　　The southern part of China is twoseason continuouscropping rice area， with high cropping index and high interaction strength between hosts and pathogens， which would cause variation of pathogenicity of pathogens easily， thereby resulting in the loss of variety resistance. At the end of 1980s， a strongly virulent pathotype appeared in Guangdong Province， while the main resistant cultivars containing gene Xa4 in south rice area and even all varieties planted in production could not resist isolates of this pathotype[13]. In the early 21st century， a pathotype IX with stronger virulence was then found in Guangdong Province， which greatly threatens rice production[9].On this basis， in this study， the disintegration and variation of the pathogenicity of Xoo in south China rice area was further investigated， so as to provide a scientific basis for the control of rice bacterial blight using variety resistance， the selection of targeted resistant gene for breeding and the rational distribution of resistant varieties. 　　Materials and Methods
　　Materials
　　Tested pathogenic isolates and rice： Diseased leaf sample were collected from different riceproducing regions in southern China， and 150 bacterial strains were isolated for the detection of pathogenicity. Two sets of differential hosts， i.e.， the Chinese differential system including IR26， Nanjing15， Java 14， Tetep and Jingang 30[6] and the international differential system with a series of nearisogenic lines （NILs） including IRBB5， IRBB13， IRBB3， IRBB14， IRBB2 and IR24 carrying different known resistance genes[10]， were used for the detection of the isolates. Meanwhile， main cultivars planted in the southern part of China including Guangdong， Guangxi and Hainan， new varieties participating in the regional tests of national rice varieties and the regional tests of Guangdong rice varieties， and candidate variety resources bred by main breeding units such as agricultural college， South China Agricultural University in recent year， 500 rice varieties in total were collected， for the detection of the pathogenic spectra of main races of Xoo in the southern part of China.
　　Medium： WPDA （Wakimoto potatodextrose agar） medium： potato 300 g， calcium nitrate tetrahydrate 0.5 g， sodium phosphate dibasic dodecahydrate 2.0 g， peptone 5.0 g， sucrose 15.0 g， agar 17.0 g， water 1 000 ml， pH 6.8-7.0. All the reagents were purchased from Sangon Biotech （Shanghai） Co.， Ltd.
　　Methods
　　Detection of pathotypes （races） of tested isolates
　　The two sets of differential hosts， Chinese differential system and the international differential system with a series of nearisogenic lines were both planted in isolated concrete ponds. Various varieties were transplanted in different rows in single plant mode according to a planting spacing of 18 cm×24 cm. At the booting stage， leaves were cut off， and the rice plants were inoculated with bacteria. The bacterial isolates were cultured on WPDA plate at 28-30 ℃ for 48-72 h， washed with sterile water， and prepared into suspensions with a concentration of 3×108 CFU/ml. Scissors were dipped with corresponding suspension and used for cutting off 2-3 cm of leaf tip by one dipping one leaf. Each strain was inoculated onto 20 leaves of rice plants in one hill of each differential host， with two replicates in random arrangement. With reference to the disease grades of isolates in China， investigation and recording were performed 21 d after inoculation. If the scab has a length shorter than 1/4 of the length of the inoculated leaf or the scab has an area smaller than 20% of the area of the inoculated leaf， the plant is resistant to the disease， and if the scab has a length longer than 1/4 of the length of the inoculated leaf or the scab has an area larger than 20% of the area of the inoculated leaf， the plant is susceptible to the plant[6]. According to the reaction modes of various isolates on the differential hosts， the strain types （Chinese differential hosts） and races （nearisogenic lines） were divided. 　　Determination of the pathogenic spectrum of main races
　　The main isolates （dominant population and highly virulent type） in the southern part of China were selected for the determination of the pathogenicity on the 500 parts of main cultivars， candidate varieties and germplasm resources in the south China rice area. For all the tested varieties， seedlings were raised in normal season to the seedling age of 30 d for early rice and 25 d for late rice. Each variety was planted in one row in single plant mode according to a planting spacing of 18 cm×24 cm. One row of resistant IRBB5 and one row of susceptible Jingang 30 were planted as the resistant and susceptible controls， respectively. In the differential nurseries， the fertilization level was generally slightly higher than open field， and banded sclerotial blight and pests were timely controlled. At the adultplant stage， artificial inoculation was performed by cutting leaves to the various varieties with the main isolates and races of bacterial blight identified in "Detection of pathotypes （races） of tested isolates"， respectively. The various isolates were cultured on WPDA plates at 26-28 ℃ for 48-72 h. Each strain was then washed off with sterile water and diluted to 6×108 CFU/ml by turbidimetry. Other inoculation methods were according to "Detection of pathotypes （races） of tested isolates". The inoculation was performed after15：00， and completely 2 h after the preparation of the used bacterial suspension. Next， 21 d after the inoculation， the scab length was measured leaf by leaf and plant by plant， and the desired mean values were recorded. The resistant and susceptible dividing value is 25% of the length of diseased leaf， i.e.， If the scab has a length shorter than 1/4 of the length of the inoculated leaf， the plant is resistant to the disease， and if the scab has a length longer than 1/4 of the length of the inoculated leaf， the plant is susceptible to the plant.
　　Analysis of disintegration and variation of the pathogenicity of Xoo
　　The detection results of the pathogenicity of the tested isolates on the Chinese differential hosts were combined with history research data， so as to the comparatively analyze the disintegration and variation dynamics of the pathogenicity of the Xoo. The research team of this study determined 450 isolates of Xoo during 1988-1993[14]， 168 isolates during 1996-2000[15] and 53 isolates in 2004 in Guangdong Province. Base on the identified pathotypes and their frequencies of occurrence， comparison was performed with the results in this study， and the evolution process and development trends of dominant pathotypes were analyzed. 　　Results and Analysis
　　Pathogenic reaction of isolates on Chinese differential hosts
　　The resistant and susceptible reaction of the tested isolates on Chinese differential hosts IR26， Nanjing 15， Java 14， Tetep and Jingang 30 included 6 pathotypes， i.e.， SRRRR （ type I）， SSRRR （type II）， SSSRR （type III）， SSSSR （type IV）， SSRRS （type V） and SSSSS （type IX）， respectively. There were 21 type I isolates， 23 type II isolates， 11 type III isolates， 29 type IV isolates， 41 type V isolates and 25 type IX isolates， accounting for 14.00%， 15.33%， 7.30%， 19.30%， 27.40% and 16.67% of tested isolates， respectively. It was indicated that type V and type IV isolates are dominant populations in the southern part of China （Table 1）.
　　Pathogenic reaction of isolates on nearisogenic lines with known resistant genes
　　The pathogenic races of the tested isolates on the nearisogenic lines with known resistant genes （IRBB5， IRBB13， IRBB3， IRBB14， IRBB2 and IR24） were seven races， i.e.， R1 （RRRRRR）， R2（RRRRRS）， R3 （RRRRSS）， R4 （RRRSSS）， R5 （RRSSSS）， R8 （RSSSSS）and R10 （RRRSRS）， respectively. The occurrence frequencies of R1， R2， R3， R4， R5， R8 and R10 were12.00%， 7.33%， 6.00%， 11.33%， 15.34%， 44.67% and 3.33%， respectively （Table 2）. Among them， R8 is a dominant race.
　　Agricultural Biotechnology2018
　　Table 1 Reaction of Chinese differential varieties to isolates of Xoo
　　Pathotype
　　Differential varieties
　　Jingang 30TetepNanjing 15Java 14IR 26
　　No. of isolatesPercentage oftotal isolates∥%
　　ISRRRR2114.00
　　IISSRRR2315.33
　　IIISSSRR117.30
　　IVSSSSR2919.30
　　VSSRRS4127.40
　　VISRSRR00.00
　　VIISRSSR00.00
　　VIIIRRRSR00.00
　　IXSSSSS2516.67
　　R： Resistant； S： Susceptible
　　Disintegration and variation dynamics of the pathogenicity of Xoo
　　The determination of Xoo during 1988-2000 exhibited five pathotypes， i.e.， I， II， III， IV and V， and the dominant pathotype was always pathotype IV. The Occurrence frequencies were in the range of 23.30%-45.16% （Table 3）. In 2004， it was found that the tested isolates showed a new reaction mode， SSSSS （IX）， on the Chinese differential hosts， Jingang 30， Tetep， Java14 and IR26， but pathotype IV was still the dominant pathotype. Only three isolates were of the new pathotype， accounting for 5.7%. The results showed that the occurrence frequency of pathotype V was higher than pathotype IV， and became the first among the various pathotypes， as well as the dominant pathotype （Fig. 1）. The proportion of pathotype IX increased from 5.70% in 2004 when it occurred for the first time to 16.67% in 2014. 　　Fig. 1 Development of major Xoo pathotypes IV， V and IX in southern China during 1988-2014
　　Table 2 Reaction of isolates of Xoo on rice nearisogenic lines
　　Pathotype
　　Rice nearisogenic lines
　　IRBB5IRBB13IRBB3IRBB14IRBB2IR24No. of strainsPercentage oftotal strains∥%
　　R1RRRRRR1812.00
　　R2RRRRRS117.33
　　R3RRRRSS96.00
　　R4RRRSSS1711.33
　　R5RRSSSS2315.34
　　R6RSRRRR00.00
　　R7RSSRRS00.00
　　R8RSSSSS6744.67
　　R9SSSSSS00.00
　　R10RRRSRS53.33
　　R： Resistant； S： Susceptible
　　Table 3 Monitoring of the variation of Xoo pathotype in southern China during 1988-2014
　　Year No. of strains
　　Percentage of pathotype∥%
　　IIIIIIVIVIX
　　19886211.2938.713.2345.161.61-
　　19899716.4931.9011.3440.210.00-
　　1990829.7630.4910.9741.466.00-
　　19915020.0026.008.0040.006.00-
　　19929820.4121.439.1838.7810.20-
　　19936116.3927.878.2036.0711.47-
　　19963016.7023.3013.3026.7020.00-
　　19972516.0024.008.0028.0024.00-
　　19984022.5020.005.0030.0022.50-
　　19993016.7020.0020.0026.7016.70-
　　20004318.6020.9016.3023.3020.90-
　　20045317.0022.6011.3024.5018.905.70
　　201415014.0015.307.3019.3027.4016.67
　　Pathotypes of main populations of Xoo in southern China
　　The pathogenic rates of the three pathotypes IV， V and IX as the main populations of Xoo in the southern part of China and pathogenic races R8 and R5 on the 500 main cultivars， candidate varieties and germplasm resources collected in the southern part of China were 50.40%， 95.00%， 96.40%， 62.00% and 42.60%， respectively. It was indicated that pathotypes IX and V has stronger virulence， and few varieties could resist these two pathotypes； R8 also has stronger virulence， and most varieties could not resist it； pathotype IV has medium to strong pathogenicity， and there are basically equal resistant and susceptible varieties； and R5 has weak to medium virulence， and most varieties could resist it （Fig. 2）.
　　Fig. 2 Pathogenicity test predominant races of Xoo in southern China
　　Discussion
　　The detection results of Chinese differential hosts showed that in 1996-2000， there were five pathotypes of Xoo in the southern part of China， i.e.， pathotypesI， II，III， IV and V， and the dominant pathotype was always pathotype IV， though its occurrence frequency decreased gradually. However， in the period， the occurrence frequency of pathotype V increased gradually， and in 2014， its occurrence frequency was detected to be higher than that of pathotype IV， i.e.， it became the dominant pathotype instead of pathotype IV in rice areas in the southern part of China. Among the pathotypes of Xoo in China， the pathotype V strains are highly virulent strains causing the loss of resistance in main cultivars in the southern part of China carrying resistant gene Xa4. The pathotype was firstly found by the research team of this study at the end of last century in Guangdong Province， which has spread from the southern part of China to the Yangtze River Basin[2，16-17]， greatly threatening the safe production of rice in China. In 2004， a strain reacting as fully susceptible （SSSSS） to the five Chinese differential hosts was found. The reaction mode is different from the reaction modes of Xoo on Chinese differential hosts reported in China， i.e.，SRRRR （pathotype I）， SSRRR （pathotype II）， SSSRR （pathotype III）， SSSSR （pathotype IV）， SSRRS （pathotype V）， SRSRR （pathotype VI）， SRSSR （pathotype VII） and RRRSR （pathotype VIII）， so it is a new pathotype[9]. The new pathotype IX has a wide pathogenic spectrum， with stronger virulence. Its occurrence frequency was only 5.7% in 2004， but in this study， the strains of this pathotype accounted for 16.67% among the various pathotypes. It is necessary to strongly alert the rapid development， and close attention should be paid to its development dynamics. Chen et al.[12]determined the 29 strains from Guangxi Province also in the southern part of China， which all showed the SSSSS strongly virulent mode on the Chinese differential hosts. The strongly virulent strains of pathotypes V and IX are all found in the southern part of China for the first time and spread to other areas of China， indicating that Xoo in the southern part of China has the characteristics of rapid variation of pathogenicity， more populations and strong virulence. 　　The bacterial strains in the southern part of China were detected with the international nearisogenic lines with known resistant genes， and it was found that there were seven pathogenic races， i.e.， R1， R2， R3， R4， R5， R8， R9 and R10， respectively. Among them， the dominant races were R8 and R5， the occurrence frequencies of which were 44.67% and 15.34%， respectively. However， Yang et al.[10] divided all the strains in China into nine races， i.e.， R1， R2， R3， R4， R5， R6， R7， R8 and R9， respectively. Among them， the dominant races were R8 and R5， respectively， the occurrence frequencies of which were 26% and 23%， respectively. The two studies differed in the pathogenic races， but the dominant races were the same， which were all both strongly virulent， indicating that R8 and R5 are the dominant races of Xoo in China. The determination of the pathotypes of the main populations of Xoo on the variety resources in the south China rice area showed that the dominant pathotype V strains and R8 race as well as the strongly virulent pathotype IX strains had higher pathogenic rates， and most of current main cultivars were not resistant to them. The pathotype V strains with strong virulence have become the dominant strains in the southern part of China， and the dominant race with strong pathogenicity and pathotype IX strains with stronger virulence developed rapidly. According to the research results of the resistance of rice to Xoo[18-20]， and the breeding for resistance to Xoo in the southern part of China should select the resistant genes xa5， Xa7， Xa23 and xa34.
　　This study simultaneously used Chinese differential hosts and international nearisogenic lines with known resistant genes for the determination ofthe disintegration of pathogenicity of Xoo in the southern part of China， and combined with the results of history studies and similar experimental results， the pathogenicity of the two sets of differential hosts and their variation and disintegration dynamics were compared and analyzed. Chinese differential hosts are suitable for local climate， the healthy leaves （free of inoculation） were dark green， and obviously different from the scab of inoculated leaves， so the disease could be identified easily. Furthermore， rice varieties differ in resistance and susceptibility gradients and include different Indica and Japonica types， resulting in better differential ability of strains in different rice areas in China. However， some varieties have unclear genetic background or carry multiple genes， so the correspondence between the pathotypes and resistant genes is not clear enough. Furthermore， with the occurrence of strong virulent strains， the resistance in all differential hosts has been overcome， and it is necessary to supply varieties with higher resistance. The advantage of the international nearisogenic lines with known resistant genes is the clear corresponding relationship between the pathogenic races and the resistant genes， but the hosts are all foreigner varieties， some of which have poor adaptability to the climate in China， and the healthy leaves free of inoculation often yellow physiologically with unclear disease and health conditions. In addition， the representativeness of variety resistance is also limited， and no highly susceptible genotype varieties similar to Chinese differential host Jingang 30 have been found yet， while the differential hosts must include most susceptible varieties. Therefore， the resistant genes with differential ability would be further introduced into local varieties， so as to develop the differential hosts with local genetic background. 　　References
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